Lepraric acid derivatives as chemotaxonomic markers in Hypoxylon aeruginosum, Chlorostroma subcubisporum and C. cyaninum, sp. nov

Hypoxylon aeruginosum (Xylariaceae), an infrequently encountered predominantly tropical pyrenomycete, of which two varieties are known to science, is characterised by having a cyan blue stromatal surface or subsurface. In the course of our ongoing chemotaxonomic evaluation of the Xylariaceae, specific profiles of H. aeruginosum were observed by high performance liquid chromatography, coupled with diode array detection and mass spectrometry (hplc-DAD/MS). By comparison with an authentic standard, lepraric acid and several yet unidentified metabolites with similar hplc-DAD/MS characteristics were detected in the stromata of the type material and other specimens of this species. Interestingly, lepraric acid was hitherto only known from lichenised ascomycetes. Hypoxylon aeruginosum, which is here reported first from Africa and Asia, contained none of the metabolites that were previously detected in other Xylariaceae, except for stromata growing hyperparasitically on other Hypoxylon species. A different lepraric acid derivative was also detected in the type specimen of Chlorostroma subcubisporum, which differs from H. aeruginosum by having a green stromatal surface, cuboid ascospores, and in lacking an amyloid ascal apical apparatus. A second species of Chlorostroma, which showed essentially the same metabolite profile as H. aeruginosum, is described from Thailand. We conclude that Chlorostroma and H. aeruginosum are closely related. However, no taxonomic conclusions are drawn from these findings because no cultures have so far become available to study their anamorphic morphology, their secondary metabolites in culture, and their molecular phylogeny. Taxonomic novelty: Chlorostroma cyaninum L?ss?e, Srikitikulchai & J. Fournier, sp. nov.     

<Emphasis Type="Italic">Ruwenzoria</Emphasis>, a new genus of the Xylariaceae from Central Africa

During a foray to the mountain rainforests of the Democratic Republic of the Congo, a peculiar species of Xylariaceae was found, which could not be accommodated in any of the existing genera. It is recognised as representative of a new genus, named Ruwenzoria, owing to the presence of a new combination of teleomorphic and anamorphic characters that are regarded as significant for generic segregation within the Xylariaceae. Studies on its secondary metabolites in stromata and cultures by high performance liquid chromatography, coupled with diode array and mass spectrometric detection (HPLC-DAD/MS), and on its phylogenetic affinities based on 5.8S/ITS rDNA sequence data, respectively, revealed a close relationship of the new taxon to the genera Daldinia and Entonaema, from which it differs by having neither a hollow, gelatinous nor a conspicuously zonate stromatal interior, and an anamorph featuring enteroblastic rather than holoblastic conidiogenesis. A specimen from the same geographic region, previously identified as Daldinia bakeri by R.W.G. Dennis was found to constitute a mixture of stromata of Ruwenzoria and an additional, undescribed Daldinia species. The latter fungus is not formally described due to the scantiness of the material, but its morphological characteristics are illustrated.     

Recognition of hypoxyloid and xylarioid <Emphasis Type="Italic">Entonaema</Emphasis> species and allied <Emphasis Type="Italic">Xylaria</Emphasis> species from a comparison of holomorphic morphology,HPLC profiles,and ribosomal DNA sequences

The genus Entonaema comprises Xylariaceae with hollow, gelatinous stromata that accumulate liquid. Some of its species, including the type species, appear related to Daldinia from a polyphasic approach, comprising morphological studies, comparisons of ribosomal DNA sequences, and high performance liquid chromatography (HPLC) profiles with diode array and mass spectrometric detection (HPLC-DAD-MS). This methodology was used to study Entonaema pallidum. Its major stromatal constituent was identified as xylaral, a secondary metabolite known from Xylaria polymorpha. This compound was detected in several Xylaria spp., including the tropical X. telfairii and morphologically similar taxa, whose stromata may also become hollow and filled with liquid. Cultures of E. pallidum resembled those of Xylaria, substantially differing from other Entonaema spp., in their morphology, 5.8S/ITS nrDNA sequences, and HPLC profiles. The type specimen of E. mesentericum was located in the spirit collection of the herbarium B and found to agree morphologically with the nomenclatorily younger E. pallidum. Traces of xylaral were even detected by HPLC-DAD-MS in the spirit in which the fungus had been preserved. Entonaema pallidum is thus regarded as a later synonym of E. mesentericum. Therefore, the latter name is transferred to Xylaria. A key to entonaemoid Xylariaceae is provided. Colour reactions (NH₃, KOH) of the ectostroma were applied to a limited number of Xylaria spp., but metabolite profiles of cultures appear more promising as chemotaxonomic traits to segregate this genus. As xylaral was also found in Nemania and Stilbohypoxylon spp., while being apparently absent in Hypoxylon and allied genera, it may be a chemotaxonomic marker for Xylariaceae with Geniculosporium-like anamorphs.     

Soft coral Sarcophyton (Cnidaria: Anthozoa: Octocorallia) species diversity and chemotypes

Research on the soft coral genus Sarcophyton extends over a wide range of fields, including marine natural products and the isolation of a number of cembranoid diterpenes. However, it is still unknown how soft corals produce this diverse array of metabolites, and the relationship between soft coral diversity and cembranoid diterpene production is not clear. In order to understand this relationship, we examined Sarcophyton specimens from Okinawa, Japan, by utilizing three methods: morphological examination of sclerites, chemotype identification, and phylogenetic examination of both Sarcophyton (utilizing mitochondrial protein-coding genes MutS homolog: msh1) and their endosymbiotic Symbiodinium spp. (utilizing nuclear internal transcribed spacer of ribosomal DNA: ITS- rDNA). Chemotypes, molecular phylogenetic clades, and sclerites of Sarcophyton trocheliophorum specimens formed a clear and distinct group, but the relationships between chemotypes, molecular phylogenetic clade types and sclerites of the most common species, Sarcophyton glaucum, was not clear. S. glaucum was divided into four clades. A characteristic chemotype was observed within one phylogenetic clade of S. glaucum. Identities of symbiotic algae Symbiodinium spp. had no apparent relation to chemotypes of Sarcophyton spp. This study demonstrates that the complex results observed for S. glaucum are due to the incomplete and complex taxonomy of this species group. Our novel method of identification should help contribute to classification and taxonomic reassessment of this diverse soft coral genus.     

Species diversity and community structure in neotropical fruit-feeding butterflies

Chemical secretions that are explicitly tied to species recognition may potentially be informative for phylogenetic reconstruction, especially when traditional morphological or molecular characters lack resolution. Anal sac secretions from 16 species within the family Felidae (order Carnivora) were chemically analysed and their utility as phylogenetic characters was assessed. Results were generally consistent across the different chemical data types (e.g. glycolipids, neutral lipids, or phospholipids). Two major clades were indicated, falling out according to body size: one for species greater than 30 kg (Panthera, Uncia, and Puma) and another for those less than 12 kg (remaining species). The primary solutions agreed with respect to the species pairs Prionailurus + Leptailurus, Caracal + Lynx, Oncifelis+Leopardus, Otocolobus+Felis, Panthera leo + P pardus, and P. tigris + Uncia. The only area of disagreement between chemical types was the positioning of the mountain lion (Puma concolor); however, this species appears to cluster with the cheetah (Acinonyx jubatus) in the Tjig cat clade. Although our solutions differ from six previously proposed hypotheses of felid phylogeny (morphological and molecular), the previous estimates all differ strongly amongst themselves reflecting the historical uncertainty regarding felid systematics. Phytogenies derived from the lipid data were very robust and decisive. Few equally most parsimonious trees were obtained, consistency indices were much higher than their expected values, and bootstrap and Bremer support values were also high. Thus, our findings illustrate the species-specific nature of chemical signals and their usefulness as phylogenetic characters.     

Rapid identification of teleocidins in fermentation broth using HPLC photodiode array and LC/MS methodology

Summary During the course of screening for inhibitors of phorbol ester binding to protein kinase C, several actinomycete cultures were discovered that produce active metabolites. HPLC coupled to photodiode array and LC/MS techniques were applied to broth extracts to identify the presence of indolactams belonging to the teleocidin family. Various members of this family were rapidly identified from crude broth extracts using a combination of these spectroanalytical procedures. An analytical HPLC system was developed to optimize separation of teleocidin, A and B analogues directly from ethyl acetate extracts of whole broth cultures. This technique allowed us to identify a novel homologue of pendolmycin and demonstrated the utility of photodiode array HPLC coupled with LC/MS as an intial analytical tool in the analyses of these secondary metabolites produced by soil microorganisms.     

Screening and evaluation of fungal resources for loratadine metabolites

Loratadine is a selective inverse agonist of peripheral histamine H1-receptors. Microbial biotransformation gained a lot of attention for its ability to convert molecules to valuable medicinally active substances. The main objective of the present research was to investigate the ability of different fungi to biotransform the drug loratadine to its active metabolite desloratadine, because desloratadine is four times more potent, possess longer duration of action than loratadine and is effective at low doses. The screening studies were performed with selected fungi using their respective broth media and sterile incubation conditions. The drug and metabolites formed (if any) were extracted and analysed using HPLC analysis. Structural elucidation and confirmation of metabolites were by mass and proton NMR spectroscopy. Among the six fungi selected, Cunninghamella elegans, Cunninghamella echinulata and Aspergillus niger cultures showed extra peaks at 3.8, 3.6 and 4.1 min, respectively, in HPLC when compared with their controls, which indicated the formation of metabolites. The metabolites thus formed were isolated and their structures were confirmed as dihydroxy desloratadine, desethoxy loratadine and 3-hydroxy desloratadine by Cunninghamella elegans, Cunninghamella echinulata and Aspergillus niger cultures, respectively, by mass spectrometry and NMR spectroscopy. Three fungi were identified to have the ability to biotransform loratadine to its active metabolite and other different metabolites.     

中国几种剧毒鹅膏菌的ITS序列分析

测定了来自中国不同地区的9个剧毒鹅膏菌的核糖体内转录间隔区(ITS)核苷酸序列.以湖南鹅膏为外类群,用ITS序列构建了系统进化树.结果表明:两个不同产地的欧氏鹅膏存在一定的差异,但仍可聚为同一组;欧氏鹅膏与其它几种剧毒鹅膏的亲缘关系较远;两个根据形态特征鉴定为灰花纹鹅膏的标本可能是两个不同的种;欧氏鹅膏、致命鹅膏和黄盖鹅膏白色变种这3个产生白色子实体的种系统演化上不是同源的.     

The genus Pyrenomyxa and its affinities to other cleistocarpous Hypoxyloideae as inferred from morphological and chemical traits

Types and authentic specimens of Hypoxylon piceum, Pulveria porrecta, and Pyrenomyxa invocans were studied for morphological traits and extrolite (= secondary metabolite) profiles generated by analytical HPLC with UV-visual and mass spectrometric detection. The orange stromatal pigments of P. invocans are rubiginosin A and mitorubrinol. It lacks three different types of extrolites (BNT, macrocarpone and hypomiltin) that are known from Hypoxylon taxa and occur in H. piceum and P. porrecta. In agreement with morphological traits, the latter two names are regarded as synonymous and transferred to Pyrenomyxa. Another species from Eastern Russia, Pyrenomyxa morganii sp. nov., is recognized. It contains yet unidentified azaphilones besides BNT and orsellinic acid, and its culture produces 5-methylmellein and a virgariella-like anamorph. These findings suggest a close relationship of Pyrenomyxa to Hypoxylon and emphasize the utility of chemotaxonomic traits for fungal taxonomy in general. Pyrenomyxa is accepted ad interim until the phylogenetic relationships among Hypoxylon have been further evaluated by means of chemotaxonomic, morphological and molecular methods.     

Phylogeny of Betaphycus (Gigartinales,Rhodophyta) as inferred from COI sequences and morphological observations on B. philippinensis

The phylogeny of the three species that comprise the genus Betaphycus Doty in Silva, Basson and Moe and their phylogenetic relationships with other eucheumatoids are still unresolved. In this study, the utility of the mitochondrial cytochrome oxidase I (COI) marker in resolving their relationships was evaluated. Analyses of the COI sequences from Betaphycus philippinensis Doty and Betaphycus speciosus (Sonder) Doty ex Silva specimens collected from their type localities (Sorsogon, Philippines and Western Australia, respectively) revealed that the two species formed a well-supported clade distinct from Eucheuma J. Agardh and Kappaphycus Doty ex Silva. The genotyped specimens of B. philippinensis were observed to exhibit dorsal protuberances, a characteristic which has been regarded as a key diagnostic feature of Betaphycus gelatinus (Esper) Doty ex Silva. This observation raised the possibility that these two taxa are conspecific. In addition, B. philippinensis specimens were also observed to exhibit morphological features that could be used to distinguish the species from other eucheumatoids, such as the location tetrasporangial nemathecium in the thallus and the presence of apical or lateral pit connections in the tetraspores. The species referred to in the literature as “B. gelatinus” (as Eucheuma gelatinae) collected from northwestern Philippines was identified as a species of Eucheuma based on molecular and morphological evidence. The phylogenetic relationships of this species with other related eucheumatoid taxa were also determined.     

Characterization and evolution of secondary metabolites in Brazilian Vernonieae (Asteraceae) assessed by LC‐MS fingerprinting

Tribe Vernonieae are well represented in Brazil, mainly by subtribe Lychnophorinae comprising mostly perennial plants that inhabit arid lands, where species have developed a number of adaptations with recognized ecological protective functions, including secondary metabolite diversification. Recent phylogenetic studies indicate that the subtribe is monophyletic, but questions regarding lineage relationships have yet to be resolved. Phytochemical investigations have also been conducted recently and provide information on the secondary metabolite chemistry of Lychnophorinae. Chemotaxonomic studies have also been carried out. However, these phytochemical investigations are fragmentary and non‐standardized. Therefore, in this study, 15 species representing the major lineages of the subtribe were selected for phytochemical investigation and reconstruction of the ancestral states of their secondary metabolites. The main secondary metabolites of these species were detected by UHPLC‐UV‐MS in different types of extract, showing the presence of trans‐cinnamic acid derivatives, flavonoids, polyacetylenes and sesquiterpene lactones. The ancestral states of these secondary metabolites were reconstructed by parsimony and indicate the occurrence of 12 putative chemical synapomorphies. In this study, we present for the first time phytochemical and evolutionary studies based on the reconstruction of the ancestral chemical character states on a phylogenetic tree of Lychnophorinae.     

Evidence of constrained phenotypic evolution in a cryptic species complex of agamid lizards

Lineages that exhibit little morphological change over time provide a unique opportunity to explore whether nonadaptive or adaptive processes explain the conservation of morphology over evolutionary time scales. We provide the most comprehensive evaluation to date of the evolutionary processes leading to morphological similarity among species in a cryptic species complex, incorporating two agamid lizard species (Diporiphora magna and D. bilineata). Phylogenetic analysis of mitochondrial (ND2) and nuclear (RAG-1) gene regions revealed the existence of eight deeply divergent clades. Analysis of morphological data confirmed the presence of cryptic species among these clades. Alternative evolutionary hypotheses for the morphological similarity of species were tested using a combination of phylogenetic, morphological, and ecological data. Likelihood model testing of morphological data suggested a history of constrained phenotypic evolution where lineages have a tendency to return to their medial state, whereas ecological data showed support for both Brownian motion and constrained evolution. Thus, there was an overriding signature of constrained evolution influencing morphological divergence between clades. Our study illustrates the utility of using a combination of phylogenetic, morphological, and ecological data to investigate evolutionary mechanisms maintaining cryptic species.     

The skeleton flight apparatus of North American bluebirds (Sialia): Phylogenetic thrushes or functional flycatchers?

To better understand ecological traits of organisms, one can study them from two, not necessarily mutually exclusive perspectives: how the traits evolved, and their current adaptive utility. In birds, foraging behavior and associated morphological traits generally are explained by a combination of adaptive and phylogenetic predictors. The avian skeleton and more specifically, the skeletal flight apparatus is under well‐known functional and phylogenetic constraints. This is an interesting area to partition the relative contributions of adaptive correlated evolution and phylogenetic constraint to species clustering in morphological space. A prediction of convergent evolution is that nonphylogenetic morphological clustering is a characteristic of ecological similarity. We tested this using representatives of North American birds from two clades, one with a mixture of foraging modes (Turdid thrushes, solitaires, and bluebirds) and one with more canalized foraging behaviors (Tyrannid flycatchers). Nine characters on the skeletal flight apparatus from 19 species were used to characterize the morphological space and test for ecomorphological clustering. When body size and phylogeny are considered, the three bluebird species and Townsend's solitaire cluster with the ecologically similar flycatchers rather than with their phylogenetic close relatives. Furthermore, sit‐and‐wait foragers tend to exhibit relatively long distal elements and a long keel while active ground foragers have deeper keels and a longer humerus. Distal elements, expected to be relatively shorter and more bowed in the flycatchers and bluebirds, were actually longer and narrower. A reduction of distal element mass may be more important for facilitating maneuverability than surface area for insertion of wing‐rotational musculature. J. Morphol. 274:909–917, 2013. © 2013 Wiley Periodicals, Inc.     

High-performance liquid chromatographic analysis of 2-aminofluorene and its metabolites in monolayer cultures of rat hepatocytes

A high-pressure liquid chromatography method was developed to separate 2-aminofluorene and several ring-hydroxylated metabolites. The acetylated derivatives of these compounds and N-hydroxy-2-acetylaminofluorene were also separated simultaneously. HPLC analyses were performed using a Dupont Zorbax C-8 HPLC column and a solvent mixture of 0.02 M acetic acid and isopropanol. Isopropanol concentrations were increased from 27 to 100% over 45 min using a concave gradient system. Desferal mesylate was added to the aqueous component to improve the resolution of several hydroxylated arylamine metabolites. The method was used to quantitate the metabolites of 2-aminofluorene in monolayer cultures of rat hepatocytes.     

New species and reports of <Emphasis Type="Italic">Hypoxylon</Emphasis> from Argentina recognized by a polyphasic approach

A preliminary account of Hypoxylon species (Xylariaceae) from the hitherto widely unexplored “Yungas” mountain forests of Northwest Argentina is presented. Two new species are described based on extensive morphological, molecular (ITS region of rDNA, partial β-tubulin gene) and chemotaxonomic data. Hypoxylon spegazzinianum is close to H. erythrostroma, but differs by larger ascospores and a virgariella-like asexual morph. Hypoxylon calileguense resembles H. subgilvum when growing on wood, but can be distinguished by larger ascospores and a fawn to brick stromatal surface colour. Stromata found on bark have affinities to H. pelliculosum, but differ in their stromatal surface colour and conspicuous amyloid apical apparatus. In addition, nine taxa of Hypoxylon are reported for Argentina for the first time, and some details on their asexual state and stromatal secondary metabolites are reported. An updated dichotomous key for Hypoxylon species from Argentina is provided.     

Using multilocus approach to uncover cryptic diversity within Pseudotrapelus lizards from Saudi Arabia

The agamid Pseudotrapelus lizards inhabit the mountainous areas of the Arabian Peninsula and eastern North Africa. Currently six Pseudotrapelus species are recognised, though diagnostic morphological characters are still lacking, creating great difficulty in describing new species. Recently, two specimens of Pseudotrapelus were collected from the vicinity of Riyadh in central Saudi Arabia, an area that was not sampled in previous phylogenetic studies. In here we used both mitochondrial and nuclear data to investigate the phylogenetic position of the new samples, and assess their phylogenetic relationships with the other recognised species of Pseudotrapelus from across the distribution range of the genus. We used a multilocus approach of haplotype networks, concatenated datasets and species trees, performed mitochondrial and nuclear species delimitation analyses, and estimated divergence times. In general, our results support previous molecular studies and uncover the presence of cryptic diversity within Pseudotrapelus. The phylogenetic structure of the genus is of two major clades and within them seven distinct, delimited phylogenetic groups belonging to the six recognised species and the seventh to the individuals from Riyadh. The Riyadh specimens were distinct in all analyses performed. We suggest that the new specimens from the Riyadh area are a distinct lineage, forming a clade with their phylogenetic relatives, P. sinaitus and P. chlodnickii. The clade formed by these three species diverged during the Late Miocene around 6.4 Ma, with cladogenesis possibly facilitated by vicariance and isolation caused due to climatic fluctuations and the progression of sandy areas. Our results suggest further morphological research is necessary to revise the taxonomic status of this lineage and of the entire genus.     

Challenges in the delimitation of morphologically similar species: a case study of <Emphasis Type="Italic">Tuber brumale</Emphasis> agg. (Ascomycota,Pezizales)

Tuber brumale (winter truffle) is one of the most controversial true truffles, not only in regard to its ecological and economical role but also its taxonomy. Multilocus phylogenetic analyses have revealed that specimens identified earlier as T. brumale belong to two species. These species were deemed cryptic right away, because preliminary morphological measurements did not show any phenotypical differences. In this study, we measured the morphology of 119 T. brumale agg. specimens, identified by DNA-based phylogenetic tools. We found several continuous morphological characters which show strong statistical differences between the two species, albeit not without overlap. Using a combination of these characters, we show that efficient separation of the two species is possible. We describe T. cryptobrumale sp. nov. and present the environmental demands and the potential area reconstruction of both species. We argue that non-representative sampling is a major culprit in most failures to detect both the existence of morphologically similar species and their morphological differences. Our findings illustrate the benefits of integrative taxonomy: the use of a combination of molecular, morphological and ecological tools.     

Evolutionary relationships within the Neotropical, eusporangiate fern genus Danaea (Marattiaceae)

Genera within the eusporangiate fern family Marattiaceae have long been neglected in taxonomic and systematic studies. Here we present the first phylogenetic hypothesis of relationships within the exclusively Neotropical genus Danaea based on a sampling of 60 specimens representing 31 species from various Neotropical sites. We used DNA sequence data from three plastid regions (atpB, rbcL, and trnL-F), morphological characters from both herbarium specimens and live plants observed in the field, and geographical and ecological information to examine evolutionary patterns. Eleven representatives of five other marattioid genera (Angiopteris, Archangiopteris, Christensenia, Macroglossum, and Marattia) were used to root the topology. We identified three well-supported clades within Danaea that are consistent with morphological characters: the "leprieurii" clade (containing species traditionally associated with the name D. elliptica), the "nodosa" clade (containing all species traditionally associated with the name D. nodosa), and the "alata" clade (containing all other species). All three clades are geographically and ecologically widely distributed, but subclades within them show various distribution patterns. Our phylogenetic hypothesis provides a robust framework within which broad questions related to the morphology, taxonomy, biogeography, evolution, and ecology of these ferns can be addressed.     

Review: Microbial synthesis of agrochemical metabolites

The synthesis of agrochemical metabolite reference standards by microbial cultures can serve as a useful alternative to conventional chemical synthesis, particularly when the chemical synthesis is difficult. Microbially generated metabolites of agrochemicals can also be useful for predicting degradative pathways in animals, plants and soils prior to conducting animal, plant and soil metabolism studies which are required by regulatory agencies to support agrochemical registrations. Examples from the literature are used to illustrate the utility of synthesizing metabolites of agrochemicals by common microbes. Received 17 January 1997/ Accepted in revised form 20 March 1997     

Assessing phylogenetic resolution among mitochondrial, nuclear, and morphological datasets in Nothonotus darters (Teleostei: Percidae)

External morphological characters are the basis of our understanding of diversity and species relationships in many darter clades. The past decade has seen the publication of many studies utilizing mtDNA sequence data to investigate darter phylogenetics, but only recently have nuclear genes been used to investigate darter relationships. Despite a long tradition of use in darter systematics few studies have examined the phylogenetic utility of external morphological characters in estimating relationships among species in darter clades. We present DNA sequence data from the mitochondrial cytochrome b (cytb) gene, the nuclear encoded S7 intron 1, and discretely coded external morphological characters for all 20 species in the darter clade Nothonotus. Bayesian phylogenetic analyses result in phylogenies that are in broad agreement with previous studies. The cytb gene tree is well resolved, while the nuclear S7 gene tree lacks phylogenetic resolution, node support, and is characterized by a lack of reciprocal monophyly for many of the Nothonotus species. The phylogenies resulting from analysis of the morphological dataset lack resolution, but nodes present are found in the cytb and S7 gene trees. The highest resolution and node support is found in the Bayesian combined data phylogeny. Based on our results we propose continued exploration of the phylogenetic utility of external morphological characters in other darter clades. Given the extensive lack of reciprocal monophyly of species observed in the S7 gene tree we predict that nuclear gene sequences may have limited utility in intraspecific phylogeographic studies of Nothonotus darters.